Purified exosome products, method of making, and methods of using

Abstract

To provide a purified product of spherical or spheroid exosomes.SOLUTION: A method of preparing a purified exosome product comprises obtaining a material comprising blood, a blood product or a non-blood product. The non-blood product includes umbilical cord Wharton's jelly, stromal vascular fraction from fat, apheresis bone marrow products, synovial fluid, cerebrospinal fluid, or mesenchymal stem cells. The method also comprises: performing filtering or apheresis of the material; pooling the material; agitating the material; under conditions effective to maintain a spherical or spheroid structure of exosomes in the material; freezing the agitated material under conditions effective to maintain the spherical or spheroid structure of the exosomes in the material; thawing the frozen agitated material at a controlled rate from 0.1°C to 5°C per minute; and lyophilizing the agitated material comprising freezing the agitated material at a controlled rate.SELECTED DRAWING: None

Description

[Technical field]

[0001] This disclosure claims priority to U.S. Provisional Patent Application No. 62 / 598,765, filed December 14, 2017, which is incorporated by reference in its entirety. Summary of the Invention

[0002] In one aspect, the disclosure describes a purified exosome product. In some embodiments, the purified exosome product comprises spherical or spheroidal exosomes having a diameter of 300 nm or less. In some embodiments, the purified exosome product comprises a population of exosomes where at least 95% of the exosomes have a diameter within a distribution range of 100 nm. In some of these embodiments, the purified exosome product comprises a population of exosomes where at least 90% of the exosomes have a diameter within a distribution range of 60 nm.

[0003] In some embodiments, the purified exosome product has a water content of less than 10%.

[0004] In some embodiments, the purified exosome product has a shelf life of at least six months without refrigeration.

[0005] In another aspect, the disclosure describes a reconstituted product, wherein any of the embodiments of the purified exosome product outlined above are reconstituted in water, hi some embodiments, the purified exosome product is provided at a concentration of 30% or less.

[0006] In some embodiments, the purified exosome product is CD63 + Exosomes and CD63 - In some of these embodiments, the purified exosome product may contain a mixture of exosomes. -In another embodiment, the purified exosome product contains between 1% and 20% CD63 - Exosomes and 80% to 99% CD63 + It may contain exosomes.

[0007] In another aspect, the disclosure generally describes a composition comprising a biocompatible matrix and any embodiment of a purified exosome product as outlined above, hi some embodiments, the biocompatible matrix can comprise collagen, thrombin, gelatin, alginate, or another natural basement membrane product.

[0008] In another aspect, the disclosure describes a method for preparing a purified exosome product. In general, the method includes obtaining a starting material, filtering the starting material, pooling the filtered material, mixing the pooled material, and lyophilizing the pooled and mixed material. The starting material can include blood, a blood product, or a non-blood product. Suitable non-blood products include, for example, umbilical cord Wharton's jelly, adipose-derived stromal vascular fraction, apheresis bone marrow products, synovial fluid, cerebrospinal fluid, or mesenchymal stem cells.

[0009] In some embodiments, the starting material is obtained from humans under the age of 30, post-operative donors, premenopausal women, perinatal women, or placenta.

[0010] In some embodiments, the method includes freezing the pooled and agitated material and thawing the pooled and agitated frozen material prior to lyophilizing the pooled and agitated material.

[0011] In some embodiments, the material is freeze-dried for at least 5 hours. In some of these embodiments, the pooled and stirred material is freeze-dried for 170 hours.

[0012] In another aspect, the present disclosure describes a method for preparing an artificial blood product, which generally involves reconstituting any embodiment of the purified exosome product outlined above in a pharma- ceutically acceptable carrier.

[0013] In some embodiments, the reconstituted blood product may be prepared by mixing the purified exosome product with a biodegradable polymer scaffold, a non-biodegradable polymer scaffold, or nanotubes.

[0014] In another aspect, the disclosure describes a method for accelerating wound healing. Generally, the method comprises administering to a wound any embodiment of an artificial blood product as outlined above in an amount effective to cause the wound to heal in a shorter time than the wound would heal in the untreated state.

[0015] In another aspect, the disclosure describes a method for increasing vascularization of a wound bed. Generally, the method comprises administering to the wound any embodiment of the artificial blood product outlined above in an amount effective to heal the wound in a shorter time than the wound would heal in the untreated state.

[0016] In another aspect, the disclosure describes a method for increasing epithelialization of a wound. In general, the method comprises administering to the wound any embodiment of the artificial blood product outlined above in an amount effective to heal the wound in a shorter time than the wound would heal in the untreated state.

[0017] In another aspect, the disclosure describes a method for inhibiting a neoplasm in a tissue. Generally, the method comprises inhibiting at least 50% of CD63 - The present invention includes administering an embodiment of an artificial blood product comprising exosomes to tissue exhibiting a neoplasm.

[0018] The above summary is not intended to describe each disclosed aspect or every implementation of the present invention. The following description more particularly illustrates exemplary embodiments. In some places in this application, guidance is provided through a series of examples, which examples can be used in various combinations. In each example, the listed list serves only as a representative group and should not be interpreted as a limiting list. [Brief description of the drawings]

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0020] [Figure 1] Diagram of purified exosome product (PEP). (A) Preparation of PEP and collagen bioscaffold gel. (B) Collagen fibers act as delivery vessels for PEP microvesicles.

[0021] [Diagram 2] Field emission scanning electron microscopy (Fe-SEM) images of collagen scaffolds with different concentrations of purified exosome product (PEP): (A) collagen only; (B) collagen with 5% PEP; (C) collagen with 10% PEP; (D) collagen with 20% PEP.

[0022] [Diagram 3] Atomic force microscopy images showing platelet-rich plasma at different magnifications (A, B) and purified exosomes (PEP) at different magnifications (C, D).

[0023] [Figure 4]In vitro cell migration scratch assay. (A) Analysis of PEP-treated, PRP-treated and untreated (FBS) human dermal fibroblasts (HDFs) at 0, 12, 24 and 48 hours shows increased migration rate with PEP treatment; pink margin depicts the scratch area. (B) Line graph representation of the imaged data in (A). (C) Quantification of wound confluence percentage by IncuCyte Essen BioScience is shown, demonstrating greater cell growth rate in bioscaffold-treated HDFs.

[0024] [Diagram 5] PEP stimulation of angiogenesis in vitro. (A) Co-cultures of NHDF and HUVEC cells were seeded in PEP, PRP or FBS on days 0 and 8. (B) Representative masked images of stimulated angiogenic networks after 8 days. Data are shown as mean ± SEM (n=8) representative of two separate experiments. Scale bar is 800 μm.

[0025] [Figure 6] Effect of PEP on Wound Closure. (A) Representative images of wound closure at days 0 and 28 of a 28-day in vivo ischemic rabbit ear study. (B) Tracking of wound bed closure for each treatment group over 28 days in vivo. (C) Data are presented as mean ± std. Statistical significance was determined using Student's t-test (***=p<0.0001 and **p<0.01). (D) Quantification of wound size demonstrated that biogel closed faster compared to collagen-treated and untreated wounds.

[0026] [Figure 7]Histological analysis of ischemic wound healing 2 weeks after surgery. (A and B) Representative H&E images are shown for each treatment: nonischemic control, ischemic untreated control, ischemic wound with collagen addition, and ischemic wound with PEP addition. Scale bar: 1 mm; 20x magnification. (C and D) Ischemic wounds on rabbit ears show significant cellular infiltration and increased epidermal thickness at the wound edges. H&E, hematoxylin and eosin.

[0027] [Figure 8] α-SMA immunohistochemical staining of cells in the wound bed 28 days after surgery. Myofibroblasts differentiated from fibroblasts are indicated by arrows. α-SMA positive cells around newly formed blood vessels are indicated by arrow heads. *=p-value<0.01.

[0028] [Figure 9] PEP-induced skeletal muscle growth. PEP induces rapid proliferation of myoblast precursors (MyoD+ satellite / myoblasts). Altering culture conditions with PEP induced myotube formation in culture (actinin).

[0029] [Figure 10] Skeletal muscle proliferation by PEP. PEP induces rapid proliferation of myoblast precursors after 24 and 48 hours (right panel) relative to standard culture conditions (FBS). Altering culture conditions with PEP induces myotube formation in culture (PEP 48 hours).

[0030] [Figure 11] Wound repair with PEP. (A) The use of PEP in the subcutaneous space in muscle injuries induced a significant increase in cellular abundance within the surgical collagen scaffold over a period of as long as 2 weeks, which was not observed in collagen scaffolds alone. (B) After a 4-week observation period, progenitor cells differentiated into either skeletal muscle or adipose tissue (depending on the proximity to similar tissues) within PEP-loaded scaffolds, whereas collagen-only scaffolds remained decellularized.

[0031] [Figure 12]Western blot analysis to detect proteins contained within PEP preparations with activity in suppressing oxidative stress. Three different batches of PEP (B2, B3, B4) were dissolved in 20% solution (5 mL saline in a PEP vial), filtered through a 0.2 micron filter, and protein concentration was quantified using a BCA assay kit (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA). Hereby, 1.5 μL of each sample was dissolved in 23.5 μL of lysis buffer and heated at 85°C for 3 min. 20 g of protein was loaded onto a 12.5% ​​polyacrylamide gel (CRITERION, Bio-Rad Laboratories, Inc., Hercules, CA).

[0032] [Figure 13] Analysis of cell proliferation as a function of time after treatment with the indicated amounts of sorted CD63+PEP exosomes or CD63-PEP exosomes. CD63+PEP exosomes promoted continued cell proliferation over the duration of the study compared to the negative control (serum-free medium). CD63+PEP exosomes promoted further continued proliferation after approximately 30 hours, whereas the positive control showed rapid proliferation for approximately 20 hours and then plateaued or grew slowly. CD63-PEP exosomes inhibited cell proliferation compared to the negative control. The presence of both of these populations within the PEP allows for the appropriate induction of cell proliferation in response to treatment, while preventing uncontrolled proliferation.

[0033] [Figure 14]Analysis of PEP on extracellular vesicles (EVs) or exosomes derived from alternative traditional methods. Ultracentrifugation and tangential flow filtration are two established methods for enriching exosomes or EVs from solution. Here, nanosite-based analysis of exosome size and quantity revealed a highly heterogeneous population of EVs when these methodologies were performed with sizes >200 nm and particle numbers well below 1×1010 / mL. On the other hand, the present PEP-derived methodology results in a very narrow size range of exosomes (<100 nm) and particle yields in excess of 1×1010.

[0034] [Figure 15] Intracellular delivery of PEP. Immunofluorescence of red fluorescently tagged PEP exosomes demonstrated rapid uptake of this exosomal product into cells.

[0035] [Figure 16] Delivery of fluorescently labeled PEP to the ischemia-reperfusion site reveals rapid uptake due to capillary leakage. Labeled PEP with a far-red fluorescent dye was delivered to pig hearts 10 min after 90 min of relief of infarction in the left anterior descending limb. Macroscopic analysis of pig hearts via the Xenogen System shows the presence of far-red fluorescent dye in the infarcted region. Histological analysis shows the presence of p-selectin (an exosome marker) in the infarcted region versus the absence of p-selectin in non-infarcted regions. This demonstrated that PEP has the ability to exploit capillary leakage after injury for embedding in tissues, e.g., myocardium.

[0036] [Figure 17] Biodistribution of PEP labeled with a far-red fluorescent dye, as visualized by the Xenogen System, was seen to target the liver when given IV and the gastrointestinal tract when given IP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure describes novel purified exosome products (PEPs) having unique structures, compositions containing the PEPs, methods for preparing the PEPs, and methods for using the PEPs. The methods of use include various applications related to wound healing. Appropriate amounts of PEPs can restore healing of otherwise damaged wounds.

[0038] Despite the nomenclature of PEP (purified exosome product), the products described herein may be prepared from extracellular vesicles and / or exomeres. Thus, unless otherwise specified herein, the term "exosomes" in this disclosure includes not only exosomes, but also exomeres and extracellular vesicles, so long as the product itself has the physical, structural, and / or functional characteristics described for PEP.

[0039] Non-healing wounds in patients with pathophysiological disorders such as diabetes, peripheral vascular disease or infections represent a major global medical problem. The complex process of wound healing is governed by multiple biological and molecular events occurring during inflammation, proliferation and extracellular matrix deposition.

[0040] In normal wounds, inflammatory cells, keratinocytes, fibroblasts, production of growth factors, cell proliferation and angiogenesis orchestrate the progression of the healing process. Inappropriate production of growth factors, reduced angiogenesis and impaired cell migration are factors that impede the normal repair process of wounds. Indeed, impaired skin perfusion due to nearby arterial occlusion, vascular compression or microvascular obstruction or thrombosis remains a central risk factor for non-healing wounds.

[0041] Current clinical treatment of non-healing wounds includes topical treatment with debridement and appropriate wound coverage. Support for wound healing may include negative pressure wound therapy (NPWT) and hyperbaric oxygen therapy (HBO). Restoration of angiogenesis may reverse the breakdown in the wound healing cycle and contribute to a sustained rate of repair. Local administration of essential wound healing growth factors by application of platelet-rich plasma (PRP) also contributes to successful repair in various models of tissue repair.

[0042] Furthermore, improvement and promotion of diabetic wounds may be achieved using cell-derived exosomes. Exosome-based therapeutic effects to accelerate wound healing and angiogenesis have been demonstrated using exosomes derived from body fluids.

[0043] Exosomes are tiny vesicles (40-100 nm in diameter) that are secreted by all different cell types and provide intercellular communication signals. A variety of different carrier molecules, including miRNA and proteins, can be transferred between cells via exosomes. Current knowledge of the function of exosomes in wound healing remains limited.

[0044] This disclosure describes novel exosome compositions, their preparation, and various applications for their use. In the studies described herein, multiple exosome preparations were evaluated to identify them at the ultrastructural level. Using this approach, novel purified exosome products (PEPs) were produced with unique ultrastructural structures.

[0045] The starting material for the preparation of the PEP may be whole blood or any suitable blood product, including, without limitation, any suitable apheresis blood product, including leukoreduced products, plasma separation products, cryoanemic plasma, fresh frozen plasma, pheresis platelet products, platelet rich plasma, platelet poor plasma, or any red blood cell and leukoreduced product. The blood or blood product may be obtained from any suitable source, including, but not limited to, the general population under 30 years of age, the general population under 40 years of age, post-operative populations, premenopausal women, perinatal women, placenta or umbilical cord blood. The starting material for the preparation of the PEP may be an alternative, suitable non-blood source, such as umbilical cord Wharton's jelly, adipose-derived stromal vascular fraction, apheresis bone marrow product, synovial fluid, cerebrospinal fluid, mesenchymal stem cells, epithelial cells, naturally occurring stem cells, embryonic stem cells, induced pluripotent stem cells, or conditioned media of these or any other cell source.

[0046] If necessary, the starting materials may be frozen until needed to prepare the PEP. Typically, the starting materials may be stored at -20°C or -80°C, and preferably in a Current Good Manufacturing Practice (CGMP) facility.

[0047] The process for the preparation of PEP begins with a filtration step. The starting material is thawed if necessary prior to filtration, for example 2 to 30 units (typically 5 to 15) of blood product. Gravity-based filtration is sufficient, but any suitable filtration step may be performed. The filtered products are pooled for product binding with several agitation steps. Any agitation method used for sufficient sample mixing may be used. Agitation may include, for example, 5 minutes of manual agitation and / or 5 to 15 minutes of mechanical agitation, but are not limited to these options. The pooled filtered products may be frozen at -20°C to -80°C if desired and stored until ready for further processing. If stored frozen, the material may be thawed under controlled conditions, for example, by warming at a rate of 0.1°C to 5°C per minute.

[0048] If desired, the filtered product may be dispensed, for example, into glass vials. Depending on the desired water content level, volumes as small as 0.1 mL to 10 mL may be used with vials as small as 1 mL and as large as 50 mL. The aliquots of product then undergo a controlled temperature change to ensure consistent lyophilization properties.

[0049] Lyophilization can be performed at any temperature below the freezing temperature of water at the atmospheric pressure (either natural or artificial) where the lyophilization is performed. Thus, in some embodiments, lyophilization can be performed at a minimum temperature of -180°C or higher, -160°C or higher, -140°C or higher, -120°C or higher, -100°C or higher, -90°C or higher, -80°C or higher, -70°C or higher, -60°C or higher, -50°C or higher, -40°C or higher, -30°C or higher, or -20°C or higher. In some embodiments, lyophilization can be performed at a maximum temperature of 0°C or lower, -5°C or lower, -10°C or lower, -15°C or lower, -20°C or lower, -25°C or lower, -30°C or lower, -35°C or lower, -40°C or lower, -45°C or lower, -50°C or lower, -55°C or lower, -60°C or lower, -65°C or lower, -70°C or lower, or -75°C or lower. In some embodiments, lyophilization may be performed within a temperature range characterized by an endpoint defined by any minimum temperature described above and any maximum temperature described above that is warmer than the minimum temperature. Thus, for example, in some embodiments, lyophilization may be performed at a temperature of -10° C. to -100° C. In the initial freezing step, the temperature may be reduced by as fast as 2° C. and as slow as 0.1° C. per minute until the desired final temperature is reached.

[0050] Once the desired final temperature is reached, a vacuum pressure is applied for primary drying. The vacuum pressure may be any suitable vacuum pressure. Thus, in some embodiments, the minimum vacuum pressure applied may be 1 mTorr or more, e.g., 5 mTorr or more, 10 mTorr or more, 15 mTorr or more, 20 mTorr or more, 25 mTorr or more, 50 mTorr or more, 75 mTorr or more, 100 mTorr or more, 150 mTorr or more, 200 mTorr or more. In some embodiments, the maximum applied vacuum pressure may be 500 mTorr or less, e.g., 400 mTorr or less, 300 mTorr or less, 200 mTorr or less, 100 mTorr or less, 90 mTorr or less, 80 mTorr or less, 70 mTorr or less, 60 mTorr or less, or 50 mTorr or less. In some embodiments, the applied vacuum pressure may be characterized as a range having endpoints defined by any minimum vacuum pressure described above and any maximum vacuum pressure greater than the minimum vacuum pressure. Thus, for example, the applied vacuum pressure may range from 10 mTorr to 300 mTorr.

[0051] This initial stage may be held for a minimum hold time of at least 15 minutes, e.g., at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 120 minutes, at least 140 minutes, at least 160 minutes, at least 180 minutes, at least 200 minutes, at least 220 minutes, or at least 240 minutes. This initial stage may be held for a maximum hold time of 30 days or less, e.g., 15 days or less, 10 days or less, 5 days or less, 1 day or less, 1200 minutes or less, 900 minutes or less, 600 minutes or less, 300 minutes or less, 270 minutes or less, 240 minutes or less, 210 minutes or less, 180 minutes or less, 150 minutes or less, 120 minutes or less, 90 minutes or less, 75 minutes or less, 60 minutes or less, or 45 minutes or less. In some embodiments, the initial phase may include a hold time characterized as a range having endpoints defined by any minimum period described above and any maximum period greater than the minimum period described above, e.g., in some embodiments, the initial phase may include a hold time of 30 minutes to 300 minutes.

[0052] Depending on the starting volume, additional drying steps and changes in final temperature are desired. For any additional drying step, the final temperature can be any temperature below the freezing temperature of water at the atmospheric pressure (either natural or artificial) where the lyophilization is performed. Suitable final temperatures are similar to those for the primary drying step described above. If more than one drying step is included in the lyophilization process, the final temperature of each drying step may be determined independently of the final temperature of the primary drying step and / or independently of any additional drying steps. In some embodiments, the additional drying steps may be performed at a temperature of -10°C to -100°C. In other embodiments, the additional drying steps may be performed at a temperature of -20°C to -140°C.

[0053] When more than one drying step is included in the freeze-drying process, the vacuum pressure for each drying step may be determined independently of the vacuum pressure of the primary drying step and / or independently of any additional drying steps. In some embodiments, the vacuum pressure for the additional drying steps may range from 10 mTorr to 300 mTorr. In other embodiments, the vacuum pressure for the additional drying steps may range from 50 mTorr to 400 mTorr.

[0054] When one or more drying steps are included in the freeze-drying process, the hold time of each drying step may be determined independently of the hold time of the primary drying step and / or independently of any additional drying steps. In some embodiments, the hold time of the additional drying steps may range from 30 minutes to 300 minutes. In other embodiments, the hold time of the additional drying steps may range from 200 minutes to 7,200 minutes.

[0055] In some embodiments, a warmer temperature drying step may be desired as well. This warmer temperature drying step may be carried out at a temperature ranging from 0° C. to 42° C. under vacuum. The vacuum pressure may be as described above for any additional drying step. The warmer temperature drying step may be carried out for any time suitable to achieve a moisture content level of 10% or less. In some embodiments, achieving such a moisture content level may take from 30 minutes to 7,200 minutes depending on the temperature and vacuum pressure conditions.

[0056] Therefore, optimal lyophilization parameters are based, at least in part, on the capacity of the equipment utilized, the moisture content of the starting material, the starting volume, and the density of the starting material (eg, serological material versus culture medium).

[0057] To arrive at a lyophilized product, a caking agent may be used for certain applications but is not necessary to derive the PEP. Suitable caking agents include, but are not limited to, polyvinylpyrrolidone (PVP), glucose, glycine, and non-crystalline sugars (e.g., sucrose, trehalose, mannitol). In some embodiments, the lyophilization process can take as little as 5 hours and as long as 170 hours. The final product after this process is visually released based on the formation of a caked solid, with release criteria requiring greater than 95% proper cake per production lot. If these metrics are not met, the entire lot is discarded.

[0058] The PEP has a different structure than conventional exosomes prepared using conventional techniques. Conventional enriched exosomes exhibit a snowflake-like structure (irrespective of freeze-drying conditions) as shown in Figures 3A and 3B. In contrast, the PEP exosomes herein are smaller and more spherical as shown in Figures 3C and 3D. Atomic force microscopy and SEM showed that the derived PEP product was highly spherical and there was no exosome aggregation as opposed to the "snowflake" like aggregated exosome structures seen in processes involving any shear forces, filtration or centrifugation.

[0059] Thus, in some cases, the PEP exosomes are distinguished from conventional exosome products by being non-crystalline in structure and spherical or spheroidal with a diameter of 300 nm or less. PEP exosomes may therefore have a maximum diameter of 300 nm or less, e.g., 250 nm or less, 200 nm or less, 175 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 95 nm or less, 90 nm or less, 85 nm or less, 80 nm or less, 75 nm or less. PEP exosomes may have a minimum diameter of at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least 55 nm, at least 60 nm, at least 65 nm, at least 70 nm, or at least 80 nm. In some cases, the diameter of PEP exosomes may be expressed as a range having an endpoint defined by any minimum diameter described above and any maximum diameter described above that is greater than the minimum diameter. In some embodiments, the PEP may therefore be characterized as having a diameter of between 50 nm and 200 nm, for example between 100 nm and 200 nm.

[0060] Additionally, Figure 14 provides data showing that the PEP preparations described herein can have a narrower diameter distribution compared to conventional exosome preparations, as described in more detail below. For example, in some embodiments, the diameter of the exosomes in the PEP preparation can have a distribution of less than 300 nm, i.e., the difference between the largest and smallest diameters. Figure 14 shows a conventional exosome preparation with a diameter distribution of 600 nm or more. Figure 14 shows a PEP preparation in which more than 95% of the exosomes have a diameter that falls within a 100 nm distribution between a diameter of 100 nm and a diameter of 200 nm, and 90% of the exosomes have a diameter that falls within a 60 nm distribution (132 nm ± 30 nm).

[0061] In some embodiments, the PEP can have a low moisture content, such as a moisture content of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

[0062] The PEP may be formulated and / or reconstituted with a pharma- ceutically acceptable carrier to form a therapeutic composition. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, diluent, isotonicity agent, physiological buffer, carrier solvent, suspension, colloid, water, and the like. The use of such media and / or agents for pharma- ceutically active substances is well known in the art. Any conventional media or agent is contemplated for use in a therapeutic composition, so long as it is not incompatible with the active ingredient. Supplementary active ingredients may be incorporated into the composition. As used herein, "pharma- ceutically acceptable" means a non-biological or otherwise undesirable material, i.e., the material may be administered to an individual together with the PEP without exerting any undesirable biological effects or interacting adversely with any other components of the therapeutic composition in which it is included. Exemplary pharma-ceutically acceptable carriers include, for example, physiological buffers, sterile water, biodegradable polymers, synthetic polymers, or basement membrane solutions of any suitable concentration. Further suitable carriers for PEP include any material capable of changing state from liquid to solid under temperature, pressure or other environmental changes, in which case PEP will dissolve in such material in the liquid phase and become entrapped within the material once solid, as shown in the examples of Figures 2 and 3 herein where the carrier is collagen.

[0063] The PEP may therefore be formulated into a therapeutic composition. The therapeutic composition may be formulated in various formats to accommodate the preferred route of administration. Thus, the therapeutic composition may be administered via known routes, for example, orally, parenterally (e.g., intradermally, transdermally, subcutaneously, intramuscularly, intraarterially, intracoronary, intravenously, intraperitoneally, etc.), or topically (e.g., intranasally, intrapulmonary, intramammary, intravaginally, intradermally, transdermally, rectally, etc.). The therapeutic composition may be administered by administration (e.g., by spray or aerosol) to mucosal surfaces, for example, the nasal or respiratory mucosa. The composition may also be administered via sustained or delayed release. In addition, the PEP, either in solution form or in combination with the above matrices / gels, may be surgically implanted into various tissues or body cavities. For reconstructive, dental, or cosmetic applications, the PEP may be delivered in liquid form or in combination with a matrix, for example, subcutaneously, submucosally, or to deeper layers of the face.

[0064] Thus, the PEP may be provided in any suitable form, including but not limited to, a solution, suspension, emulsion, spray, aerosol, or any mixture. The composition may be delivered in a formulation containing any pharma- ceutically acceptable excipient, carrier, or solvent. For example, the formulation may be delivered in a conventional topical form, such as a cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, etc. The formulation may further include one or more additives, such as adjuvants, skin penetration enhancers, thickeners, etc. In addition, the use of PEP may be applied in combination with abrasive processes, such as microdermabrasion, microneedling, laser peel, chemical peel, or other skin abrasive platforms. In these settings, the PEP may be delivered either in solution, in the base, or as a matrix / gel. Furthermore, in hair restoration, the PEP may be delivered via supradermal or subcutaneous delivery.

[0065] The formulations may conveniently be presented in unit dosage form and may be prepared by methods well known in the pharmaceutical art. Methods of preparing compositions with pharma-ceutically acceptable carriers include the step of bringing the PEP into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations may be prepared by uniformly and / or intimately bringing into association the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0066] PEP may be combined with other excipients that may modulate the structural behavior of the reconstituted / rehydrated exosome product. Suitable excipients include biological matrices, including, for example, collagen, thrombin, gelatin, alginate, or any other natural basement membrane product, applied either in mixture or purified form (including decellularized tissue scaffolds). Suitable excipients also include, for example, hyaluronic acid or thrombin adhesives to promote rapid aggregation to fill surgical or fistula formation defects. PEP is compatible with additives or excipients that have the ability to change state from liquid to solid under temperature, pressure, or other environmental changes.

[0067] As described above, the PEP may be provided in a formulation with low water content. In some embodiments, the PEP formulation may therefore have a shelf life of at least 6 months and up to 4 years without refrigeration. The PEP formulation may therefore be particularly suitable for use in, for example, underdeveloped areas or military use where wound healing is required but refrigeration is not possible, inconvenient or costly. The low water content PEP formulation easily redissolves to form a reconstituted PEP product. For example, a dried PEP formulation may redissolve as up to a 20% solution within 5 minutes. A reconstituted 20% PEP solution may form a gel over 1 hour at 37°C. The gel formulation may, for example, facilitate localization of the PEP after administration to a subject and / or create structural elements that facilitate the regenerative effects of the PEP in tissues in need of repair. Combining PEP with collagen may increase the rate at which the reconstituted PEP forms a gel at 37°C. Indeed, the rate at which the reconstituted PEP gels in the presence of collagen is at least partially influenced by the concentration of collagen. Increased rates of gelation can be achieved by using higher concentrations of collagen, with the maximum concentration being 10 mg / mL. In some embodiments, PEP is used in combination with collagen at a collagen concentration of about 5 mg / mL. It is used in combination with other gelling agents, such as thrombin glue (e.g., TISSEEL, Baxter Healthcare Corp., Deerfield, IL), hyaluronic acid, polyvinyl alcohol (PVA), polylactic-co-glycolic acid (PLGA), and others. When PEP is formulated as a gel with collagen, the PEP exosomes can attach to collagen fibrils, creating a "beads-on-a-string" appearance. In addition, depending on the osmolarity of the dissolving solution, other SEM features can be seen with PEP, including exosomes stacked on strings, spikes on strings, or flowered patterns on strings. This effect depends, at least in part, on the type and concentration of collagen in the purified collagen solution, and is more common, for example, when using PEP solutions having a concentration of about 5% to about 30%.

[0068] Whether bound to collagen or in solution, the PEP exosomes show no evidence of secondary aggregation of more than 10% to 20% PEP exosomes. Furthermore, there is no evidence of aggregates containing more than 3 exosomes. As noted above, the only exception to this is when PEP is rehydrated with a hyperosmolar solution, such as TISSEEL's CaCl2 solution.

[0069] The amount of PEP administered may vary depending on various factors, including but not limited to, the weight, health condition, and / or age of the subject, and / or the route of administration. Therefore, the absolute amount of PEP contained in a given unit dosage form may vary widely and depends on factors such as the race, age, weight and physical condition of the subject, and / or the method of administration. Therefore, it is not practical to generally state what constitutes an amount of PEP that is effective for all possible uses. However, those skilled in the art can easily determine the appropriate amount with due consideration of such factors.

[0070] In some embodiments, the method can include administering to a subject PEP exosomes having a PEP concentration in solution of at least 0.5% and up to 100%. The PEP exosomes can be administered to a subject at a minimum concentration of at least 1%, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50%. The PEP exosomes may be administered to the subject at a maximum concentration of 100% or less, e.g., 75% or less, 50% or less, 25% or less, 20% or less, 19% or more, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less. In some embodiments, the PEP exosomes may be administered to the subject at a dose within a range having an endpoint defined by any minimum concentration above and any maximum concentration above that is greater than the minimum concentration. Thus, for example, the PEP exosomes may be delivered to the subject at a concentration of at least 1% to 30% or less, e.g., at least 5% to 20% or less.

[0071] In some embodiments, the PEP may be administered, for example, from a single dose to multiple doses per week, although in some embodiments, the method may be carried out by administering PEP at a frequency outside this range.Due to the wide range of applications for which administration of PEP compositions is useful, it is not practical to specify a dosing regimen for each application.In some embodiments, the PEP may be administered from about once a month to about 5 times a week.For example, a single dose may be sufficient for the treatment of myocardial infarction.For other applications, such as wound healing, cosmetic applications, hair regrowth, weekly or daily administration may be preferred.

[0072] The PEP compositions and formulations described herein have many uses. PEP can, for example, increase the growth of mesenchymal stem cells (MSCs) and / or dermal fibroblasts to a greater extent than conventional therapies (e.g., platelet lysates) or fetal bovine serum. Similarly, PEP can induce osteogenic, chondrogenic, and / or adipogenic differentiation to a greater extent than conventional therapies (e.g., platelet lysates) or fetal bovine serum. PEP can also maintain the growth of myoblasts to a greater extent than conventional therapies (e.g., platelet lysates) or fetal bovine serum. PEP can be used to enhance the proliferation properties of cells used for immunotherapy, such as, but not limited to, CAR-T, TRuC-T, NK-CAR, and hematopoietic stem cells.

[0073] For example, PEP compositions and formulations can induce a wide range of cellular responses, mainly focused on proliferation, anti-apoptosis, immune regulation and new blood vessel formation. In the presence of PEP, damaged tissues have a tendency towards regeneration. This response is embodied in observations demonstrating enhanced expression of transforming growth factor beta (TGF-β, e.g., 50 pg / mL to 200 ng / mL depending on the PEP exosome concentration in solution), vascular endothelial growth factor (VEGF, e.g., 10 pg / mL to 2 ng / mL depending on the PEP exosome concentration in solution), epidermal growth factor (EFG, e.g., 500 pg / mL to 50 ng / mL depending on the PEP exosome concentration in solution), fibroblast growth factor (FGF, e.g., 5 pg / mL to 1 ng / mL depending on the PEP exosome concentration in solution), HGF (50 pg / mL to 200 ng / mL depending on the exosome concentration in solution), and PDGF (all subtypes including AA, BB, AB at concentrations spanning between 5 pg / mL to 300 ng / mL depending on the exosome concentration in solution). Although the response is not limited to these factors, the observation that these factors are induced in different tissues is an indication of the regenerative influence of PEP.

[0074] PEP treatment increases wound confluence via in vitro scratch assay

[0075] The effect of PEP on human dermal fibroblast (HDF) migration was compared to platelet-rich plasma (PRP) treatment using an in vitro scratch assay, previously described as the gold standard for cell migration studies (Liang et al., 2007. Nat Protoc 2:329-333). Treatment with PEP caused an increase in the migration rate of HDFs compared to control and PRP-treated fibroblasts (Figure 4). Accordingly, quantitative measurements of the scratch assay demonstrated a higher rate of both wound infiltration (Figure 4B) and wound confluence (Figure 4C) in the PEP-treated HDF condition compared to control and PRP-treated HDFs at 48 hours. Following the scratch assay, wound confluence of PEP-treated HDFs was 98% at 24 hours and over 100% at 48 hours, compared to control HDFs, which were 57% at 24 hours and 81% at 48 hours.

[0076] PEP treatment increases tube length

[0077] The basement membrane matrix tube formation assay can be used to study angiogenic signaling pathways. Indeed, PEP-treated human umbilical vein endothelial cells (HUVECs) showed rapid basal levels of tube formation, suggesting optimal conditions for wound bed vascularization (Figure 5). Quantification of total network length resulted in >15,000 microns (p-value p<0.001) in PEP growth conditions compared to 5000 microns in controls (Figure 5B). Interestingly, PRP treatment also modestly increased tube formation by 10,000 microns (p<0.001). Taken together, these results indicate that PEP treatment increases the rates of migration, proliferation, and tube formation in vitro that correlate with known standards of re-epithelialization and vascular density in vivo.

[0078] Postoperative appearance of ischemic wounds and wound healing time

[0079] A minimally invasive rabbit ear model of ischemic wound healing (Chien, S. & Wilhelmi, BJ, 2012. J Vis Exp, e3341; Chien, S., 2007. Wound Repair Regen 15:928-935) was used to assess vascular ligation. Ischemic wounds were treated with either PEP or collagen alone for 4 weeks (one treatment per week) and the extent of wound healing was compared to nonischemic and untreated ischemic wounds (Figure 6A). Quantification of wound size demonstrated that PEP-treated wounds had faster closure compared to collagen-treated and untreated ischemic wounds (Figure 6A, 6B). Specifically, 2-cm PEP-treated wounds were reduced to 0.05 mm after 28 days compared with 0.67 mm for collagen-treated wounds; nonischemic wounds were reduced to 0.06 mm after 28 days, and untreated ischemic wounds were reduced to 1.3 mm after 28 days (Figures 6A, 6B). Treatment with either collagen or PEP resulted in accelerated epithelialization at 4 weeks, as demonstrated by keratin 14 staining (Figures 7A, 7B). Therefore, PEP treatment accelerates wound closure and epithelial cell migration in an in vivo ischemic model.

[0080] Morphometric assessment of the endothelial marker vWF

[0081] Wound samples stained with sheep polyclonal vWF antibody show higher vWF stained cells in wounds treated with biogels (including PEP-collagen scaffolds shown in Figure 2) when comparing untreated controls with collagen-treated wounds or when PEP is reconstituted as a 10-20% solution to rehydrate dry collagen scaffolds (see Figures 6 and 7, PEP-rich collagen scaffolds improve wound healing to that seen in non-ischemic wounds). Quantitative analysis confirmed a statistically significant difference (p<0.01) between treated biogel populations (53.7% endothelial cells per field; n=3) and untreated populations (22.2% endothelial cells per field; n=3), corresponding to increased angiogenesis.

[0082] α-SMA expression in wound sites treated with PEP

[0083] Figure 8 shows the expression levels of α-SMA in wound sites treated with PEP for 28 days. The spindle-shaped α-SMA positive cells shown in the PEP-treated wound beds demonstrate the differentiation of fibroblasts into myofibroblasts (Figure 8). In addition, the expression of α-SMA in smooth muscle cells surrounding newly formed blood cells (arrowheads in Figure 8) indicates the onset of angiogenesis in these wound beds. Quantitative analysis of α-SMA positive cells confirmed a statistically significant difference (p<0.01) between the PEP-treated and collagen-treated and untreated populations, consistent with fibroblast differentiation and a high density of mature blood vessels.

[0084] Induction of new tissues from satellite cells

[0085] FIG. 9 shows phase contrast, immunofluorescence, and schematic diagrams of smooth muscle satellite cells grown in the presence of PEP. Here, low confluence of satellite cells demonstrates their ability to proliferate into myoblasts, myocytes (MyoD+), and ultimately organize into functional myotubes (actinin+). In FIG. 10, these findings are further supported by direct contrast of PEP with alternative growth conditions in which satellite cells do not give rise to functional tissue, such as FBS (or PRP and platelet lysate, not shown). In FIG. 11, further validation of this paradigm is provided by in vivo studies in which either collagen matrix implantation alone or followed by PEP enrichment. FIG. 11A and FIG. 11B demonstrate that in PEP-rich conditions, there is strong evidence of skeletal muscle development at 2 weeks and restoration of full-thickness muscle mass at 8 weeks. This regenerative response was not seen in the control (collagen only) population. These data provide a supporting rationale for the use of a PEP-rich environment to induce regeneration of precursor-rich tissues, such as smooth muscle-containing tissues including skin, skeletal muscle (including pharynx / larynx, urinary and anal sphincters, and diaphragm along with those associated with the musculoskeletal system), gastrointestinal tract, vagina, bladder, uterus, and other structural tissues.

[0086] Figure 12 shows Western blot analysis of three different PEP preparations, labeled with B2, B3, and B4. Each sample was probed with antibodies that specifically bind to representative exosomal proteins tubulin, superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), superoxide dismutase 3 (SOD3), CD63, heme oxygenase (HO-1), and programmed cell death ligand 1 (PD-L1). Tubulin is a ubiquitous protein in human cells. SOD1, SOD2, and SOD3 are antioxidant enzymes that limit damage caused by reactive oxygen species (ROS). CD63 is a peripheral exosomal membrane protein. HO-1 catalyzes the degradation of heme and is an enzyme that is induced by oxidative stress. PD-L1 is a transmembrane protein involved in the suppression of the immune system during certain events, such as pregnancy, tissue allotransplantation, and autoimmune diseases. FIG. 12 shows that the process used to prepare PEP produces highly consistent protein profiles, as evidenced by representative protein banding across three independently prepared PEP preparations.

[0087] Figure 13 shows the time-dependent cell proliferation using various concentrations of the PEP preparation. Figure 13 also shows the effect of sorting the PEP preparation based on CD63 expression on the surface of exosomes within the preparation. PEP preparations typically express CD63 + Exosomes and CD63 - Contains a mixture of exosomes. CD63 + Exosomes may be sorted from the PEP preparation by any method suitable for sorting membrane-bound vesicles. + Representative methods for sorting exosomes include, but are not limited to, affinity separation, magnetic bead separation, flow separation, etc. The positive control shows cell proliferation when treated with mock vesicles. Cell proliferation increases rapidly until 20 hours and then plateaus as the cells reach confluence. In contrast, cells treated with the PEP preparation continue to grow (e.g., from 20 hours to 60 hours). CD63 +Cells treated with PEP containing exosomes also show continued growth at a more or less constant rate after about 40 hours. - Cells treated with PEP carrying exosomes also show a more or less constant proliferation rate after about 40 hours, but at a lower rate than the negative control (cells growing in serum-free medium). Therefore, it is possible to suppress the proliferation of CD63 cells, as may be desired in applications involving wound healing and / or tissue regeneration. + PEP containing exosomes can promote cell proliferation. However, uncontrolled cell proliferation leads to the development of neoplasms. CD63 - PEP containing exosomes may induce cellular mechanisms that slow growth, for example by reaching confluence, and therefore CD63 + PEP preparations containing exosomes may limit the risk of unrestrained cell proliferation.

[0088] Unmodified PEP preparations, i.e., PEP preparations whose characteristics have not been altered by sorting or isolation of the exosome population within the preparation, express CD63 + and CD63 - Naturally contains a mixture of exosomes. CD63 - Exosomes can inhibit unlimited cell proliferation, and naturally express CD63 + and CD63 - Unmodified PEP preparations containing exosomes can both stimulate cell proliferation and / or tissue regeneration for wound healing and limit uncontrolled cell proliferation. - Exosomes can inhibit unlimited cell proliferation, so CD63 - PEP preparations that are enriched for exosomes, e.g., CD63 + Sorting and removal of at least a portion of the exosomes can be used as an anti-tumor therapy.

[0089] In addition, CD63 + Exosome sorting reveals that CD63 + Exosomes were removed from the native PEP preparation and then expressed as CD63 +Adding back the desired amount of exosomes allowed the PEP product to express CD63. + Exosomes and CD63 - In some embodiments, the PEP preparation contains CD63 - It may only have exosomes.

[0090] In another embodiment, the PEP preparation is + Exosomes and CD63 - Exosomes may contain both CD63 and CD63. + Exosomes and CD63 - The ratio of exosomes to CD63 may vary depending, at least in part, on the amount of cell expansion desired for a particular application. + / CD63 - Exosome ratio is CD63 + Desired cell proliferation induced by exosomes and CD63 achieved through cell contact inhibition - In some scenarios, for example in tissues with non-adherent cells (e.g. blood-derived components), this ratio may be adjusted to provide the appropriate balance of cell proliferation or cell inhibition for the treated tissue. Since cell-to-cell contact is not triggered in tissues with non-adherent cells, for example, CD63 is inhibited to prevent unlimited cell proliferation. + Conversely, if there is a desire to expand a clonal population of cells, e.g., in allogeneic cell-based therapy or immunotherapy, the exosome ratio may be reduced to ensure that a large population of cells can be derived from a much smaller source. + The ratio of exosomes can be increased.

[0091] Therefore, in various embodiments, CD63 in the PEP preparation + Exosomes and CD63 -The ratio of exosomes may be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, or 30:1. + Exosomes and CD63 - Formulated to contain exosomes at a 9:1 ratio.

[0092] FIG. 14 highlights the size distribution and total exosome quantity of techniques such as ultracentrifugation and tangential flow filtration (TFF) compared to the methods described herein that result in PEP. NanoSight analysis of these separate techniques shows that exosome (extracellular vesicle) quantities using either ultracentrifugation or TFF result in a broad distribution of exosome sizes ranging from 41 nm to 776 nm in ultracentrifugation and 56 nm to 829 nm in TFF. Conversely, a typical PEP derivative results in a narrower size distribution of exosomes (or extracellular vesicles) ranging from 65 nm to 280 nm, with the majority of exosomes being between 100 nm and 200 nm. Additionally, the quantity of particles per mL for both ultracentrifugation and TFF is 2×10 8 However, PEP preparations reproducibly yielded 6 × 10 11 Resulting in number of particles / mL.

[0093] Figure 15 demonstrates that PEP has the ability to rapidly enter cultured cells when stained with a fluorescent dye. Figure 16 shows that PEP also rapidly enters cells when delivered within a tissue environment. Here, PEP is delivered via an intracoronary approach in a porcine model of ischemia-reperfusion injury. In this model of myocardial infarction, an appropriately sized angioplasty balloon is used to occlude the LAD for 90 minutes. After reperfusion, PEP labeled with a far-red fluorescent lipid dye was injected into the left anterior descending artery. Hearts were harvested within 30 minutes of PEP delivery, and far-infrared signal was assessed macroscopically. As seen in the generated Xenogen images, all delivered PEP was trapped within the infarcted region of the heart (top panel). Histological analysis demonstrated the presence of PEP within myocardial cells in infarcted, but not non-infarcted tissues, as tracked by p-selectin (an exosome marker). This demonstrates that PEP has the ability to exploit capillary leakage after injury to rapidly embed within tissue, e.g., myocardial cells. Furthermore, this provides a rationale for intra-arterial delivery of PEP either in wound situations or to prevent tissue damage.

[0094] Figure 17 tracks the biodistribution of PEP when delivered intravenously (IV) and intraperitoneally (IP). Over observation periods ranging from 10 minutes to 6 days, nearly all PEP was distributed in the liver when delivered IV, with some signal in the spleen. IP delivery sequestered PEP in the gastrointestinal tract with no liver involvement and minimal spleen involvement. IV delivery via the retro-orbital (RO) did not show significant differences in biodistribution. This indicates that PEP can be delivered IV in health and disease when targeting the liver is desired.

[0095] Therefore, the present disclosure describes novel exosome-based therapeutics and exosome-based therapeutic compositions. The biological effects of the described exosome-based compositions are higher than conventional exosome preparations. Small ultrastructural differences between conventional exosome preparations compared to the exosome compositions described herein affect cellular uptake and utilization, and therefore the different structural compositions leading to different effects. Atomic force microscopy (AFM) studies show that our novel purified exosome product (PEP) does not form clusters and aggregates. This was not the case with platelet-rich plasma (PRP), which tends to form ice crystals or flower patterns (Figure 3). PEP was significantly functionally superior to fetal bovine serum (FBS) or other preparations of conventional purified exosomes for wound healing, wound bed vascularization, and wound re-epithelialization. Thus, isolation and purification of exosomal products as described herein, i.e. in a manner that ensures a homogenous, single ultrastructural composition, as opposed to the formation of tertiary structures, results in a dramatic upregulation in biological efficacy as shown in vitro, and culminates when complexed with collagen in a biologically effective matrix that can induce regeneration of non-healing wound beds back to that seen in non-ischemic wounds.

[0096] Conventional techniques for wound healing and / or tissue regeneration promotion may be limited by the size of the tissue to be treated. Tissue satellites will be arranged as poles of regenerative tissue spaced approximately 3 mm to 5 mm apart. The eastern tissue pole is approximately 50 μm to 500 μm and may be prepared clinically by physical dissection of a small amount of excised healthy tissue adjacent to the diseased or damaged area. PEP preparations in combination with biocompatible supports (e.g., biocompatible webs, biocompatible matrices, biocompatible scaffolds, etc.) can overcome this limitation for tissue regeneration by providing multiple "satellite" nuclei of tissue growth. Each satellite nucleus may include a biocompatible support to which the PEP preparation adheres, adheres, or is otherwise attached. The satellite nuclei (clusters of cells or tissue) may further include additional growth factors. When multiple satellite nuclei are used, the composition of each satellite nucleus may be independently designed, similar to or different from any other satellite nucleus. In use, the satellite nuclei can be positioned within the damaged tissue according to where tissue regeneration is desired. The satellite nuclei can be spaced 3mm to 5mm apart. The satellite nuclei can serve as foci of tissue regeneration occurring in parallel between the various nuclei until the tissue regenerating from the various satellite nuclei coalesces to form a continuous regenerating tissue. Figure 9 shows an example of how cell or tissue satellites can result in confluence of skeletal muscle tissue in a PEP setting.

[0097] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements or the combination of any two or more of the listed elements; the term "comprising" and variations thereof are construed as open-ended, i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one; the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 is 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0098] In the foregoing description, certain embodiments may be described in isolation for clarity, and unless otherwise specified, it is expressly stated that the features of a particular embodiment are not consistent with the features of another embodiment, and an embodiment may include any combination of compatible features described herein in connection with one or more embodiments.

[0099] For any method disclosed herein that includes discrete steps, the steps may be performed in any possible order and, where appropriate, any combination of two or more steps may be performed simultaneously.

[0100] The present invention is illustrated in the following examples, it being understood that the specific examples, materials, amounts, and steps are to be construed broadly in accordance with the scope and spirit of the invention as described herein. EXAMPLES

[0101] Example 1: Production of purified exosome product (PEP)

[0102] Apheresis blood products were collected from accredited blood banks and adherence to best clinical standards was confirmed. Product units were frozen at either -20°C or -80°C and stored in a facility following current Good Manufacturing Practices.

[0103] To begin the manufacturing process, 2-30 units (typically 5-15 units) are thawed, pooled through a gravity-based filtration step using 20-40 μm filters, and several mixing steps to mix the product before being refrozen at -20°C to -80°C. The pooled product is mixed for 5 minutes with manual mixing, followed by 5-15 minutes of mechanical mixing. Under controlled conditions, the pooled product is thawed at a rate of 0.1°C to 5°C, and specific volumes are sequestered into sterile glass vials. Depending on the desired moisture level, volumes as low as 0.1 mL to 10 mL are available in vials as low as 1 mL and as high as 50 mL. The aliquots then undergo a controlled temperature ramp to ensure uniform lyophilization characteristics. The drying step can take as little as 5 hours and as long as 170 hours. The final product after this step is visually shipped based on caked pellet formation, with release criteria requiring adequate caking of 95% or greater per production lot. If these criteria are not met, the entire lot is discarded.

[0104] Fabrication of collagen-PEP scaffolds

[0105] Purified exosome product (PEP) was obtained by lyophilizing the frozen exosome-rich solution for 48 h. Collagen at a concentration of 3 mg / mL was mixed with the lyophilized PEP to reach a final concentration of 20 w / v%. The mixed solution was slowly poured into a 6 cm Petri dish and incubated at 37°C. Figure 1A shows the various steps of the PEP manufacturing process. Figure 1B is a schematic diagram illustrating the correlation between PEP and collagen fibers.

[0106] Scanning Electron Microscope (SEM)

[0107] A field emission scanning electron microscope (Hitachi S-4700, Hitachi High-Technologies, Tokyo, Japan) was used to observe the morphological characteristics of collagen (Figures 2A, 2B) and collagen-PEP (Figures 2C, 2D) scaffolds. Scaffolds were prepared in 0.1 M sodium phosphate buffer pH 7.2, 2.5% glutaraldehyde and fixed overnight. Samples were subsequently postfixed in 1% osmium tetroxide for 1 hour, dehydrated in ethanol, and supercritically dried. Dried samples were coated with gold via a sputter coater at ambient temperature. Micrographs of the scaffolds were taken, and the particle size distribution was determined using a Beckman Coulter LS 32 instrument, ranging from 0.01 mm to 1,000 mm. The average particle size was calculated by measuring the particle size of 30 particles in each of the six SEM photographs.

[0108] Atomic Force Microscope (AFM)

[0109] Atomic force microscopy was performed to investigate the morphology of collagen and biologically effective PEP. Collagen or PEP was placed on the surface of cleaved mica disks and incubated at 37°C for approximately 30 min. After incubation, the samples were washed 4-5 times with water and then dried with nitrogen gas. Nanoscale AFM images (512 × 512 pixels) were collected at room temperature using a NanoscopeIV PicoFroce Multimode AFM (Bruker Corporation, Billerica, MA) in tapping mode and analyzed using Nanoscope Analysis Version 1.40 software (Park, S. & Terzic, A, 2010. J Struct Biol 169:243-251). Representative images are shown in Figure 3.

[0110] Human dermal fibroblast (HDF) migration assay:

[0111] HDFs were seeded into 96-well INCUCYTE IMAGELOCK tissue culture plates (EssenBioScience, Inc., Ann Arbor, MI) at 2 × 10 cells per well. 4 The cells were seeded and cultured in a humidified 37°C, 5% CO2 incubator. After 24 hours, an INCUCYTE WOUNDMAKER was used to create precise and reproducible wounds in all wells of a 96-well IMAGELOCK plate. After wounding; the culture medium was aspirated from each well, and the wells were gently washed twice with culture medium to prevent the removed cells from settling and reattaching. After washing with PBS buffer, 100 μL of culture medium was replaced with a solution of PEP diluted in DMEM (without FBS) at a concentration of 5 w / v% to determine the effect of PEP on HDF migration. The cells subsequently cultured in DMEM (with FBS) were considered as control. After incubation, the experimental plate was placed on the INCUCYTE ZOOM, and the system was allowed to equilibrate for 5 minutes. Repeated scans (every 3 hours for 48 hours) were scheduled in the ZOOM software, and images were taken and recorded. The first scan in the time course was used to generate an initial scratch mask (a digital mask showing the boundaries / leading edges of migrating cells and damage-free areas). This initial scratch mask was used for the subsequent quantification steps. Scratch masks were also calculated for all subsequent imaging time points after the first scan. In addition, a statistical analysis of HDF migration was performed; we measured the Relative Wound Density (RWD), which relies on the initial scratch mask to distinguish between cell-occupied and cell-free areas of the images. The results are shown in Figure 4.

[0112] Angiogenesis test (in vitro tube formation)

[0113] PrimeKit-Cryo (Essen BioScience, Inc., Ann Arbor, MI) was used to perform in vitro angiogenesis and tube formation. On day 0, NHDFs (Normal Human Fibroblasts) were thawed, rinsed, and plated in seeding medium in Corning 96-well plates. The NHDFs were then incubated at room temperature in a tissue culture hood for 1 hour to allow their attachment to the plate. After seeding with HUVEC CytoLight Green, the plate was incubated at room temperature for 1 hour before being placed in the INCUCYTE for imaging. The cell density for PrimeKit was optimized to meet our strict quality control guidelines for experimental performance. After seeding, the co-cultures were placed in an INCUCYTE S3, and images were automatically acquired in both phase contrast and fluorescence every 3 hours for 8 days using Tiled Field of View (FOV) mosaic imaging mode. In this mode, a total of 6 images (3 images horizontally × 2 images vertically) were acquired per well and merged into a single larger image covering nearly 50% of the well (Figure 5A). On day 1, the seeding medium was replaced with 150 μL of growth medium (provided in the kit) per well. On day 2, the test reagent (5% PEP or 5% RPR or 10% FBS) was added to the test medium. On days 4 and 7, the test reagent was replaced with fresh test reagent medium. The progress was monitored for 8 days; tube formation was dynamically processed using an extended INCUCYTE algorithm.

[0114] Animal model and rabbit surgery

[0115] Under general anesthesia and using aseptic technique, rabbit ears were prepared and the hair was neatly trimmed using surgical clippers. Ischemic wounds were then created as previously described (Ahn, ST & Mustoe, TA, 1990. Ann Plast Surg 24:17-23). ​​Briefly, an ischemic substrate was created in the wound of a rabbit ear based on closure of one or more selected segments of three layers of arteries and veins using interrupted 3-0 Nylon sutures. To create an ischemic wound, a circular, full-thickness injury was created on the ventral side of the ear with a 2-cm punch. In the experimental group, the wound was administered with the bioscaffold before applying a sterile dressing, whereas in the control group, the wound was only covered with a sterile dressing.

[0116] Organizational Diagnosis

[0117] To evaluate the cellular infiltration of the skin injury site, samples from three injuries were collected at the desired time points per group. To obtain skin samples from the biopsy area, rabbits were sacrificed and tissue was removed by dissection. The injured area of ​​skin tissue was then placed on a filter membrane (any membrane resistant to organic solvents, e.g., nitrocellulose) for stabilization, and the samples were cut exactly in half. The half wounds were either embedded directly in Optimal Cutting Temperature (OCT) tissue freezing embedding medium (for frozen sections) or fixed overnight in 4% paraformaldehyde and embedded in paraffin, so that the sectioning could start in the center of the wound. Formalin-fixed samples were cut at 8 μm and stained with hematoxylin and eosin.

[0118] Hematoxylin and eosin (H&E) staining

[0119] 8-μm paraffin sections of skin tissue were processed, cut, and dewaxed and immersed in a series of incubations in xylene (2 × 3 min), 50 xylene:50 100% ethanol (1 × 3 min), 100% ethanol (2 × 3 min), 95% ethanol (1 × 3 min), 70% ethanol (1 × 3 min), 50% ethanol (1 × 3 min), and finally H2O (1 × 5 min). Slides were stained with Harris hematoxylin solution (HHS32, Sigma-Aldrich, St. Louis, MO) for 5 min at room temperature and subsequently washed in a staining bottle under running tap water until the water ran colorless (approximately 5 min). Slides were immersed in acid alcohol (1% HCl in 70% ethanol) 2 or 3 times until the sections turned pink. Slides were washed in tap water for 3 to 5 min and then immersed in aqueous ammonia (1 mL NH4OH in 1 L H2O) 5 or 6 times until the sections darkened significantly. Slides were washed in tap water for 3 to 5 min, followed by the addition of an aqueous solution of eosin Y (HT110232, Sigma-Aldrich, St. Louis, MO) to the slides for 1 min. Slides were washed again in tap water for 3 to 5 min. Slides were then dehydrated by a series of incubations in 95% ethanol (2 × 3 min), 100% ethanol (2 × 3 min), 50 xylene:50 100% ethanol (1 × 3 min), and xylene (2 × 3 min). Slides were stored in xylene (for no more than 1 h) until they were coverslipped with Permount or a xylene-based mounting medium.

[0120] Immunohistochemistry

[0121] 8-μm paraffin sections of skin tissue were processed, cut, dewaxed, and submerged in a series of incubations in xylene (2 × 3 min), xylene:ethanol (1 × 3 min), 100% ethanol (2 × 3 min), 95% ethanol (1 × 3 min), 70% ethanol (1 × 3 min), 50% ethanol (1 × 3 min), and finally HO (1 × 5 min). Antigen retrieval was then performed by immersing the tissue sections in pre-warmed Tris-EDTA buffer (10 mM Tris, 1 mM EDTA pH 8) or 0.01 M citrate buffer (pH 6) in a microwave (850 W) for 3 min. Slides were heated at 360 W for 10 min. Following this, they were allowed to cool to room temperature (RT) for 30 min before being washed in 1 × PBS. Sections were incubated in PBS containing 0.1% Triton for 5 min and then washed three times with PBS for 5 min per wash. Sections were immediately subjected to a blocking step. Before incubation with the antibodies, sections were incubated with 10% goat serum, 1% BSA, 0.01% Triton diluted in PBS for 1 h at room temperature to prevent nonspecific binding of the primary antibodies. After 1 h of blocking, slides were gently tapped on blotting paper to remove the blocking solution. Primary antibodies were diluted in PBS containing 0.5% BSA, and each section was incubated with 120 μL of primary antibody at 4°C overnight in a humidity chamber. Negative controls were incubated with 0.5% BSA / PBS to remove the primary antibody. After incubation, unbound primary antibodies were removed with three PBS washes for 3 min per wash. Each slide was then incubated with 120 μL of poly-HRP-anti-mouse / rabbit / rat IgG for 1 h at room temperature.

[0122] After washing with PBS (3 x 5 min), coverslips were mounted with Mowie Oil and left to harden at room temperature. Results were visualized and photographed with a Leica confocal microscope.

[0123] Confocal microscopy

[0124] All images were taken with a Leica TCS-SP5 confocal microscope at 40x or 20x magnification. The excitation lasers for fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) dyes were standardized for each experiment, with DAPI dye set visually for each image as required. Image processing was performed using PHOTOSHOP 7.0 imaging software (Adobe System Inc, San Jose, CA).

[0125] Example 2

[0126] Three different batches of PEP were dissolved in 20% solution (5 mL saline in a PEP vial), filtered through a 0.2 micron filter, and protein concentration was quantified using a BCA assay kit (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA). From this, 1.5 μL of each sample was dissolved in 23.5 μL of lysis buffer and warmed at 85°C for 3 min. 20 g of protein was loaded onto a 12.5% ​​polyacrylamide gel (CRITERION, Bio-Rad Laboratories, Inc, Hercules, CA).

[0127] The results are shown in Figure 12.

[0128] Example 3

[0129] Milteni CD63 magnetic beads were utilized to separate positive and negative exosome populations. These populations were precipitated and quantified prior to serial dilution for culture-based evaluation. In the IncuSite system, cultured HUVECs were placed in 5% PEP (positive control), serum-free solution (negative control), and the indicated CD63+ / CD63- concentrations. Results are shown in Figure 13.

[0130] Example 4

[0131] The exosome population was purified using ultracentrifugation at 30,000×g for 16 hours and tangential flow filtration using a 50KDa mass separation filter for the PEP derivation step. The liquid form samples from ultracentrifugation and TFF were diluted 1000-fold and loaded into the NanoSight for analysis. Lyophilized PEP was dissolved in sterile water as a 100% solution and diluted 1000-fold before evaluation with the NanoSight system (the gold standard for EV characterization) for size distribution and quantification. The results are shown in FIG. 14.

[0132] Example 5

[0133] RFP and far-red range fluorescent lipid dyes were added to the 20% PEP preparation and centrifuged at 17,000 g for 10 min to wash out unbound dye. The resuspended pellet was sonicated for homogenization and filtered through a 0.2 μm filter to remove aggregates prior to delivery into cell culture conditions (FIG. 15), intracoronary delivery following myocardial infarction (FIG. 16), and IV delivery for biodistribution analysis (FIG. 17).

[0134] The complete disclosures of all patents, patent applications, and publications cited herein, and electromagnetically available materials (including, for example, nucleotide sequence entries in Genbank and RefSeq, and amino acid sequence entries in, for example, SwissProt, PIR, PRF, PDB, and translations of annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. In the event of any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall govern. The withholding of detailed descriptions and examples is given only for clarity of understanding. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, but variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0135] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be construed in all circumstances as modified by the term "about." Accordingly, unless specifically indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by ordinary rounding techniques.

[0136] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​setting forth the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their corresponding testing measurements.

[0137] Unless otherwise noted, all headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading.

Claims

1. 1. A method for preparing a purified exosome product, comprising: Ingredients include: blood, blood products, or Non-blood products, including: Umbilical cord Wharton's jelly, Adipose-derived stromal vascular fraction Apheresis bone marrow product synovial fluid cerebrospinal fluid, or Mesenchymal stem cells filtration or apheresis; a pool of said material; agitating the material; freezing the stirring material; thawing the frozen agitated material at a controlled rate of 0.1°C to 5°C per minute; and freeze-drying the agitation material, including freezing the agitation material at a controlled rate.

2. 10. The method of claim 1, wherein the material is obtained from a human under 30 years of age, a post-operative donor, a premenopausal woman, a perinatal woman, or a placenta.

3. The method of claim 1 , wherein the material comprises umbilical cord blood.

4. The method of any one of claims 1 to 3, further comprising freezing the material at a temperature of -20°C or below.

5. 5. The method of any one of claims 1 to 4, wherein filtering the starting material comprises using gravity-based filtration.

6. 6. The method of any one of claims 1 to 5, further comprising isolating the pooled material prior to freeze-drying.

7. 7. The method of any one of claims 1 to 6, wherein the pooled and agitated material is freeze-dried for at least 5 hours.

8. 8. The method of claim 7, wherein the pooled and agitated material is freeze-dried for up to 170 hours.

9. 9. The method of any one of claims 1 to 8, wherein the material comprises blood from a premenopausal woman.

10. 10. The method of any one of claims 1 to 9, wherein the material comprises blood from a perinatal woman.

11. The method of any one of claims 1 to 10, wherein the material comprises placenta-derived blood.

12. The method of any one of claims 1 to 11, wherein the material comprises blood from an umbilical cord.

13. 13. The method of any one of claims 1 to 12, wherein the material comprises umbilical cord-derived jelly.

14. 14. The method of any one of claims 1 to 13, wherein freeze-drying the agitation material comprises cooling the frozen agitation material at a controlled rate of 0.1°C to 2°C per minute.

15. The method according to any one of claims 1 to 14, wherein a portion of the spherical or spheroid exosomes have a diameter of 300 nm or less.

16. 1. A method for preparing an artificial blood product, comprising: Preparation of a purified exosome product by the method of any one of claims 1 to 15, and Reconstituting the purified exosome product in a pharmaceutically acceptable carrier.

17. 17. The method of claim 16, wherein the pharmaceutically acceptable carrier comprises a physiological buffer, sterile water, or basement membrane solution.

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